EP1980613A1 - Procede de maintien de la fonction des cellules de tissus du foie sur une periode prolongee - Google Patents

Procede de maintien de la fonction des cellules de tissus du foie sur une periode prolongee Download PDF

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Publication number
EP1980613A1
EP1980613A1 EP07706702A EP07706702A EP1980613A1 EP 1980613 A1 EP1980613 A1 EP 1980613A1 EP 07706702 A EP07706702 A EP 07706702A EP 07706702 A EP07706702 A EP 07706702A EP 1980613 A1 EP1980613 A1 EP 1980613A1
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Prior art keywords
tissue cells
function
liver
hepatic tissue
hepatic
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EP07706702A
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German (de)
English (en)
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EP1980613B1 (fr
EP1980613A4 (fr
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Masayuki Yamato
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Tokyo Womens Medical University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers

Definitions

  • the present invention relates to a method of maintaining the function of liver tissue cells useful in the fields of biology, medicine, pharmacy and the like for a long period of time and a method for producing liver tissue cells for transplantation which enables long-term maintenance of the function of liver tissue cells to be used in the method.
  • the liver is an central organ of the metabolic function such as glyconeogenesis, storage of glycogen, lipid metabolism, production of plasma proteins, bilirubin metabolism, hormone metabolism and vitamin metabolism, and additionally has complicated functions of performing the production of bile, detoxication by various enzymes and biophylaxis against foreign substances as well.
  • a disease such as hepatitis, liver cirrhosis and liver cancers appears in the organ, the living body with the above described damaged function is remarkably adversely affected. And, when the disease proceeds, suitable measures to act for such functions have to be taken.
  • Japanese Patent Publication A No. H05-192138 describes a method of culturing dermal cells comprising culturing dermal cells on a cell culture support where the surface of a base material is coated with a polymer having an upper or lower critical dissolution temperature in water of 0 to 80°C at a temperature below the upper dissolution critical temperature or above the lower critical dissolution temperature and thereafter regulating the culturing temperature to a temperature above the upper limit dissolution temperature or below the lower limit critical dissolution temperature to peel the cultured dermal cells.
  • the present invention is made to intend to solve the problems of the above described conventional techniques.
  • the present invention has an object to provide a novel method of maintaining the function of hepatic tissue cells over a long period of time based on a completely different way of thinking from the conventional techniques.
  • the present inventors has made an examination from various angles to carry out research and development.
  • the functions of hepatic tissue cells can be maintained for a long period of time by culturing hepatic tissue cells on a cell culture support whose surface is coated with a polymer which shows a change in the hydration force in the temperature range of 0 to 80°C within the temperature range where the hydration force of the polymer is weak, thereafter changing the temperature of the liquid culture medium to a level at which the hydration force of the polymer becomes strong to peel cultured hepatic tissue cells, and then transplanting the obtained hepatic tissue cells into a definite site in vivo.
  • the hepatic tissue cells transplanted into the living body have the function of an artificial liver.
  • the present invention is completed on the basis of this founding.
  • the present invention provides a method of maintaining the function of hepatic tissue cells over a long period of time comprising culturing hepatic tissue cells on a cell culture support whose surface is coated with a polymer which shows a change in the hydration force in the temperature range of 0 to 80°C within the temperature range where the hydration force of the polymer is weak, thereafter changing the temperature of the liquid culture medium to a level at which the hydration force of the polymer becomes strong to peel cultured hepatic tissue cells, and then transplanting the obtained hepatic tissue cells into a definite site in vivo.
  • the present invention provides an artificial liver utilizing the method of maintaining the function of hepatic tissue cells over a long period of time.
  • the present invention provides an animal other than a human in which hepatic tissue cells have been transplanted utilizing the method of maintaining the function of hepatic tissue cells over a long period of time.
  • the present invention provides a system of evaluating the function of the liver comprising administering a test substance to an animal transplanted with hepatic tissue cells and judging the influence of the test substance on the function of the liver.
  • the present invention provides a method for producing hepatic tissue cells for transplantation which enables long-term maintenance of the function of hepatic tissue cells comprising culturing liver tissue cells on a cell culture support whose surface is coated with a polymer which shows a change in the hydration force in the temperature range of 0 to 80°C within the temperature range where the hydration force of the polymer is weak and thereafter changing the temperature of the liquid culture medium to a level at which the hydration force of the polymer becomes strong to peel the cultured hepatic tissue cells.
  • the method of maintaining the function of hepatic tissue cells for a long period of time described in this invention would enable long-term maintenance of the function of cultured hepatic tissue cells, would enable carrying out long-term fundamental research relating to the function of the liver as a whole, and furthermore would enable production of artificial livers and animals transplanted with hepatic tissue cells which require the expression of the function of the live over a long period of time.
  • the hepatic tissue cells which are used in the present invention are cells collected from the hepatic tissue which may contain hepatic parenchymal cells to express the function of the liver.
  • the other cells may be either nonparenchymal cells within the hepatic tissue or other cells, and include, for example, sinusoidal endothelial cells, Kupffer cells, stellate cells, pit cells, bile duct epithelial cells, vascular endothelial cells, fibroblasts and mesenchymal stem cells, and are not particularly limited. Further, these cells include cells directly collected from tissue and cell lines and their types are not limited.
  • the origin of these cells is not particularly limited and includes, for example, a rat, a mouse, a guinea pig, a marmoset, a rabbit, a dog, a cat, a sheep, a pig, a chimpanzee and an immunodeficient animal thereof, and in the case of using liver tissue cells in the treatment of humans, cells originated from a pig and a chimpanzee are desirably used.
  • the medium for cell culturing in the present invention is not particularly limited if the medium is the one conventionally used for the cells to be cultured.
  • the number of cells seeded on culturing in the present invention varies depending on the animal species of the cells used and is typically not less than 1 x 10 4 /cm 2 , preferably not less than 3 x 10 4 /cm 2 and more preferably, not less than 6 x 10 4 /cm 2 .
  • seeding concentrations of less than 1 x 10 4 /cm 2 the proliferation of hepatic tissue cells is inferior to deteriorate the extent of expression of the function of the obtained hepatic tissue cells and such concentrations are not preferred in the present invention.
  • the above described cells are cultured on a cell culture support whose surface is coated with a polymer which changes in the hydration force in the temperature range of 0 to 80°C in the temperature range where the hydration force of the polymer is weak.
  • the temperature for culturing the cells is preferably 37°C.
  • the temperature-responsive polymer which is used in the present invention may be either a homopolymer or a copolymer.
  • Such a polymer includes, for example, polymers described in Japanese Patent Publication A No. H02-211865 .
  • the polymer can be obtained by homopolymerization or copolymerization of the following monomers.
  • the monomers which can be used include, for example, a (meth)acrylamide compound, an N-(or N,N-di)alkyl substituted (meth)acrylamide derivative and a vinyl ether derivative and in the case of a copolymer, any two or more kinds of these compounds can be used.
  • copolymers with monomers other than the above described monomers, graft polymers or copolymers of the polymers with one another and mixtures of the polymers with the copolymers may be used. Further, it is possible to crosslink the polymer within the range of not damaging the inherent properties of the polymer.
  • the method of coating various kinds of polymers on the surface of a base material is not particularly limited and may follow the method described in Japanese Patent Publication A No.
  • such coating can be performed by subjecting the base material and the above described monomer or polymer to any one of electron beam (EB) irradiation, ⁇ -ray irradiation, ultraviolet ray irradiation, plasma treatment, corona treatment and organic polymerization reaction or by physical adsorption such as coating and kneading.
  • EB electron beam
  • the amount of the hydrophilic polymer immobilized at the cell adhering site may be an amount enough to move the cells and is not particularly limited, and since the cells to be used are hepatic tissue cells, their immobilization amount is not less than 0.4 ⁇ g/cm 2 , preferably not less than 0.8 ⁇ g/cm 2 and more preferably, not less than 1.0 ⁇ g/cm 2 .
  • the amount of the polymer immobilized may be measured in accordance with the conventional method which includes, for example, a method of directly measuring the cell adhered site with the use of FT-IR-ATR and a method of immobilizing a previously labeled polymer in the same manner as above described and estimating the amount of the polymer immobilized from the amount of the labeled polymer immobilized at the cell adhering site, and any method may be used.
  • the shape of the culture base material in the present invention is not particularly limited and includes, for example, a form of a dish, a multi-plate, a flask or a cell insert and a flat membrane form.
  • the base material provided with coating substances capable of generally giving a form such as a polymer compound and a ceramic, including glass, modified glass, a compound such as polystyrene and polymethyl methacrylate which are conventionally used in cell culturing all can be used.
  • the temperature-responsive polymer coated on the base material causes hydration and dehydration by changing temperature and the temperature range is found to be 0°C to 80°C, preferably 10°C to 50°C and more preferably, 20°C to 45°C. At temperatures of exceeding 80°C, there is a possibility that the hepatic tissue cells die out, and thus such temperatures are not preferred. Further, at temperatures lower than 0°C, in general, the proliferation rate of cells is extremely decreased or the hepatic tissue cells die out, and such temperatures are not preferred, either.
  • the cultured hepatic tissue cells are allowed to adhere to a carrier, if necessary, and can be peeled as they are together with the carrier by setting the temperature of the support base material to which the cells adhere at a temperature of the hydration of the coated polymer of the support base material.
  • a water flow may be applied in between the cell sheet and the support to smoothly perform peeling.
  • peeling of the sheet may be performed in the liquid culture medium where the cells have been cultured or in other isotonic solutions, and the peeling method can be selected in accordance with the object.
  • the cultured hepatic tissue cells in the present invention are not susceptible to the damage by a proteolytic enzyme represented by dispase, trypsin or the like on culturing. Therefore, the hepatic tissue cells peeled from the base material have an adherent protein and when the hepatic tissue cells are peeled in the form of a sheet, the cell-to-cell desmosome structure is to be maintained to some extent. On account of this, the hepatic tissue cell sheet can well adhere to the tissue of an affected part on transplantation and transplantation can be efficiently performed.
  • the cell-to-cell desmosome structure can be peeled while maintaining 10 to 40% thereof but a cell-to-base material basement membrane-like protein or the like is mostly destructed and accordingly, the strength of the obtained cell sheets becomes weak.
  • the hepatic tissue cell sheet of the present invention is in the state that not less than 60% of both the desmosome structure and the basement membrane-like protein remain, and various effects as described above can be obtained.
  • the sheet may be a laminate of a plurality of sheets and the number of lamination is not particularly limited. Lamination of the hepatic tissue cell sheet improves the cell density per sheet unit area to improve the function as a hepatic tissue cell sheet, and accordingly is preferred.
  • poly(N-isopropylacrylamide) has a lower limit dissolution temperature at 31°C and causes dehydration in water at a temperature of not lower than 31°C if it is in a free state and the polymer chain is coagulated to become cloudy. Inversely, at temperatures of not higher than 31°C, the polymer chain causes hydration to become in a state dissolved in water. In the present invention this polymer is coated and immobilized on the surface of a base material such as a petri dish.
  • the polymer on the surface of the base material causes dehydration in the same manner but the polymer chain is coated and immobilized on to the surface of the base material, and thus the surface of the base material shows hydrophobicity.
  • the polymer on the surface of the base material causes hydration but the polymer chain is coated and immobilized on the surface of the base material, and thus the surface of the base material is to show hydrophilicity.
  • the hydrophobic surface at this time is a suitable surface to which cells can adhere to proliferate, and further the hydrophilic surface becomes a surface to which the cells cannot adhere and the cells or the cell sheet during culturing is to be peeled only by cooling.
  • the carrier which is used in allowing hepatic tissue cells or a hepatic tissue cell sheet to adhere thereto is a structure for holding the cells of the present invention and, for example, a polymeric membrane or a structure molded from the polymeric membrane, a metallic jig or the like can be used as the carrier.
  • specific materials include, for example, polyvinylidene difluoride (PVDF), polypropylene, polyethylene, cellulose and its derivative, paper, chitin, chitosan, collagen and urethanes.
  • PVDF polyvinylidene difluoride
  • the shape of the carrier is not particularly limited.
  • the obtained hepatic tissue cells or hepatic tissue cell sheet is to be transplanted into a predetermined site within the living body.
  • the site to be transplanted may be any site within the living body and is not particularly limited.
  • the site to be transplanted includes, for example, subcutaneous tissue, intraperitoneal tissue, the liver, and a muscle.
  • the site to be transplanted may or may not be previously provided with the induction of angiogenesis and is not particularly limited.
  • the method of inducing angiogenesis is not particularly limited and includes, for example, a method of embedding FGF (fibroblast growth factor) of a blood growth factor in a microsphere and allowing the microsphere to act within the living body for 8 to 10 days while changing the composition, the size and the range of injection of this microsphere, a method of cutting a polyethylene terephthalate mesh to any size to prepare a bag, placing a high concentration agarose solution dissolving FGF in the bag and thereafter eliminating the bag after 8 to 10 days to prepare an angiogenesis induced space
  • FGF fibroblast growth factor
  • the hepatic tissue cells to be transplanted in the present invention maintain the basement membrane-like protein and have extremely good bioadhesiveness.
  • the number of cells may vary in transplantation depending on the object, and when the cells to be transplanted are in the form of a sheet, the total activity of the function of the liver can be varied by changing the size or the shape of the sheet.
  • the transplanted hepatic tissue cells or hepatic tissue cell sheet is to express the function of the liver in the human living body for a long period of time to come to an artificial liver as it is.
  • the peeled hepatic tissue cells are in the form of a sheet, the amount of expression of the function can be controlled by the size or the shape of the sheet or both.
  • Such an artificial liver is used with the object of the substantial treatment of each disease illustrated by hepatic enzyme deficiency, hemophilia, coagulopathy, liver failure, fulminant hepatitis, chronic hepatitis, liver cirrhosis, a liver resected patient and an infectious disease or assistance to the function of the liver, and its use is not particularly limited.
  • hemophilia is explained here, the deficiency of human blood coagulation factor VIII and/or human blood coagulation factor IX is the cause of the substantial onset of hemophilia, and the artificial liver of the present invention is to produce these factors to supplement them.
  • an effective gene for the production of human blood coagulation factor VIII and/or human blood coagulation factor IX may be introduced into the hepatic tissue cell sheet of the present invention as the conventional technique, and its method is not particularly limited.
  • the transplanted hepatic tissue cells or hepatic tissue sheet is to express the function of the liver within the living bodies of the animals, in other words, to come to animals transplanted with hepatic tissue cells as it is.
  • the peeled hepatic tissue cells are in the form of a sheet, the quantity of expression of the function can be controlled by the size or the shape of the sheet or both.
  • the animals which are used herein include, for example, a rat, a mouse, a guinea pig, a marmoset, a rabbit, a dog, a pig, a chimpanzee and an immunodeficient animal thereof, and are not particularly limited.
  • Such animals transplanted with hepatic tissue cells are, for example, used with the object of the system of evaluating the function of the liver by administering a test substance to this animal transplanted with hepatic tissue cells to judge the influence of the test substance on the function of the liver or the like but their use is not particularly limited thereto.
  • hepatic tissue cells (number of seeding cells: 2 x 10 4 , 37°C, 5% CO 2 ) were cultured. After three days it was confirmed that the hepatic tissue cells on the cell culture base material became confluent, and thereafter parchment paper (SS-25) was selected as a carrier for moving the cultured cells and left to stand on the cultured cell sheet. The cultured hepatic tissue cells were allowed to adhere on to the SS-25 and the cell culture base material was cooled at 20°C for 60 minutes. After cooling, the peeled cell sheet was collected from the carrier and was subcutaneously transplanted into a mouse in accordance with the method of transplanting hepatic tissue cells as shown below.
  • Fig. 1 and Fig. 2 The section of the tissue of the transplanted part 120 days after transplantation is shown in Fig. 1 and Fig. 2 .
  • the enlarged photograph in Fig. 1 is an enlargement of the inside of a rectangle in the Figure.
  • the part stained with HE, the part staining hAAT and the part staining albumin are shown in Fig. 2 , respectively.
  • the function of the liver was confirmed by measuring the murine serum hAAT concentrations in accordance with the conventional method.
  • the obtained results are shown in Fig. 3 . It can be understood that according to the technique of the present invention, the hepatic tissue cell sheet transplanted into the site having undergone the induction of angiogenesis with bFGF expresses the function of the liver over a long period opt time.
  • Example 2 The same experiment as in Example 1 was carried out except that the sustained release device obtained by dissolving bFGF in agarose was not subcutaneously inserted into the mouse and the mouse did not undergo the induction of angiogenesis. The obtained results are shown in Fig. 3 .
  • a cell culture base material poly(N-isopropylacrylamide) of a temperature-responsive polymer was coated to 1.9 ⁇ g/cm 2 , and hepatic tissue cells (number of seeding cells: 2 x 10 4 , 37°C, 5% CO 2 ) were cultured. Three days after culturing, the temperature of the culture base material was set at 20°C and cooled for 15 minutes to recover a hepatic tissue cell sheet. The hepatic cell sheet was applied to a new culture plate and was recultured to prepare a hepatic tissue sheet group ( Fig. 4a ) (Example 2).
  • the murine hepatic tissue cell sheet as obtained in Example 1 was transplanted by application into a murine subcutaneous site having undergone the induction of angiogenesis in accordance with the method of transplanting a hepatic cell sheet as shown in Example 1.
  • 3-methylcholantrene Sigma-Aldrich, St. Louis, MO
  • CYP1A of a drug metabolizing enzyme was not induced while with the prepared murine subcutaneous hepatic tissue administered with 3-methylcholantrene, CYP1A was strongly induced. It can be understood that according to the technique of the present invention, the hepatic tissue cell sheet expresses the function of the liver over a long period of time. Further, CYP1A means CYP1A activity.
  • a larger hepatic tissue sheet was prepared in accordance with the method as shown in Example 1 by laminating the murine hepatic tissue cell sheets on transplantation. Specifically, a group (mark ⁇ in Fig. 6 ) obtained by transplanting one murine hepatic tissue sheet into the murine subcutaneous site having undergone the induction of angiogenesis and a group (mark ⁇ in Fig. 6 ) obtained by transplanting the two sheets thereinto were prepared. With the two groups, the blood human alpha-1 antitrypsin concentration of the transplanted mice was measured up to 140 days after transplantation and evaluation was made by examining the stability of the transplanted tissue and the change of the amount of the tissue. It has been found that with the group transplanted with the two hepatic tissue cell sheets, a significantly large tissue was prepared compared with the group transplanted with the one sheet, and furthermore the tissue stably exists.
  • Utilization of the method of maintaining the function of hepatic tissue cells over a long time as shown in the present invention would enable long-term maintenance of the function of cultured hepatic tissue cells, would enable long-term fundamental research relating to the function of the liver and also would enable production of artificial livers and hepatic tissue cell transplanted animals which require the expression of the function of the liver over a long period of time.

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EP07706702.3A 2006-01-12 2007-01-12 Procede de maintien de la fonction des cellules de tissus du foie sur une periode prolongee Not-in-force EP1980613B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2006033057 2006-01-12
PCT/JP2007/050360 WO2007080990A1 (fr) 2006-01-12 2007-01-12 Procede de maintien de la fonction des cellules de tissus du foie sur une periode prolongee

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EP1980613A1 true EP1980613A1 (fr) 2008-10-15
EP1980613A4 EP1980613A4 (fr) 2009-05-20
EP1980613B1 EP1980613B1 (fr) 2016-01-06

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US (1) US20100239498A1 (fr)
EP (1) EP1980613B1 (fr)
JP (4) JPWO2007080990A1 (fr)
DK (1) DK1980613T3 (fr)
ES (1) ES2561944T3 (fr)
WO (1) WO2007080990A1 (fr)

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JP5320501B1 (ja) * 2012-12-10 2013-10-23 ニッカン工業株式会社 移植用細胞シートを運搬するためのキャリア
JP2015198619A (ja) * 2014-04-09 2015-11-12 大日本印刷株式会社 細胞培養容器、細胞培養装置および細胞構造体の製造方法
JP6130868B2 (ja) * 2015-02-19 2017-05-17 テルモ株式会社 単離されたシート状細胞培養物の製造方法

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KIKUCHI A ET AL: "TWO-DIMENSIONAL MANIPULATION OF CONFLUENTLY CULTURED VASCULAR ENDOTHELIAL CELLS USING TEMPERATURE-RESPONSIVE POLY(N-ISOPROPYLACRYLAMIDE)-GRAFTED SURFACES" JOURNAL OF BIOMATERIALS SCIENCE. POLYMER EDITION, VSP, UTRECHT, vol. 9, no. 12, 1 January 1998 (1998-01-01), pages 1331-1348, XP002947553 ISSN: 0920-5063 *
OKANO T ET AL: "A NOVEL RECOVERY SYSTEM FOR CULTURED CELLS USING PLASMA-TREATED POLYSTYRENE DISHES GRAFTED WITH POLY(N-ISOPROPYLACRYLAMIDE)" JOURNAL OF BIOMEDICAL MATERIALS RESEARCH, WILEY, NEW YORK, NY, US, vol. 27, no. 10, 1 October 1993 (1993-10-01), pages 1243-1251, XP008020855 ISSN: 0021-9304 *
See also references of WO2007080990A1 *
TSUDA Y ET AL: "The use of patterned dual thermoresponsive surfaces for the collective recovery as co-cultured cell sheets" BIOMATERIALS, ELSEVIER SCIENCE PUBLISHERS BV., BARKING, GB, vol. 26, no. 14, 1 May 2005 (2005-05-01), pages 1885-1893, XP025280758 ISSN: 0142-9612 [retrieved on 2005-05-01] *
YAMADA N, OKANO T, SAKAI H, KARIKUSA F, SAWASAKI Y, SAKURAI Y: "Thermo-responsive polymeric surfaces; control of attachment and detachment of cultured cells" MAKROMOL. CHEM., RAPID COMMUN., vol. 11, no. 11, March 1990 (1990-03), pages 571-576, XP002522610 *
YAMATO M, OKANO T: "Cell sheet engineering" MATERIALS TODAY, vol. 7, no. 5, April 2004 (2004-04), pages 42-47, XP002522611 *

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JP2017074050A (ja) 2017-04-20
JP2013143963A (ja) 2013-07-25
EP1980613B1 (fr) 2016-01-06
ES2561944T3 (es) 2016-03-01
EP1980613A4 (fr) 2009-05-20
JP2015204838A (ja) 2015-11-19
US20100239498A1 (en) 2010-09-23
WO2007080990A1 (fr) 2007-07-19
JPWO2007080990A1 (ja) 2009-06-11
DK1980613T3 (en) 2016-02-08

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